The nucleic acids, RNA and DNA, represent naturally occurring oligonucleotides. As used herein, the term "oligonucleotide" means homopolymer or heteropolymer sequences of nucleosides in which the nucleosides are linked with a phosphodiester bridge.
Due to advances in chemical technology, oligonucleotides comprising several hundred nucleosides or bases can now be synthetically produced. Oligonucleotide Synthesis: A Practical Approach, ed. by M. J. Gait, IRL Press, Washington, D.C. (1984). Synthetic oligonucleotides have great scientific and therapeutic utility. Synthetic oligodeoxynucleotides, for example, have widespread use in the field of recombinant DNA. Gait, supra at 1. In recent years, synthetic oligonucleotides have been shown to have therapeutic potential as antisense agents to inhibit gene expression. Uhlman, E. and Peyman, A., Chemical Reviews, 90(4): 544-583 (1990).
An antisense agent is a compound that binds to or hybridizes with a nucleotide sequence in a target nucleic acid, RNA or DNA, to inhibit the function of said target nucleic acid. Because of their ability to hybridize with both RNA and DNA, antisense agents can interfere with gene expression at the level of transcription, RNA processing or translation.
At the present time, however, the development of practical scientific and therapeutic applications of antisense technologies is hampered by a number of technical problems. See e.g.; Klausner, A., Biotechnology, 8:303-304 (1990); Armstrong, L., Business Week, Mar. 5, 1990. Such problems include (1) degradation by endogenous nucleases, (2) the high cost of production, (3) lack of sequence specific hybridization to target nucleic acids, (4) nonuniformity due to the presence of chiral phosphorous centers and (5) inadequate delivery to desired targets, for example, due to inappropriate solubility coefficients, membrane transport and cellular tracking.
One approach to preparing antisense agents that are stable, nuclease resistant, inexpensive to produce and which can be delivered to and hybridize with nucleic acid targets throughout the body is to synthesize oligonucleotide analogs having modifications in the internucleoside bridges or linkages.
As used herein, the phrase "oligonucleotide analog" refers to homopolymer or heteropolymer sequences of nucleosides or analogs thereof with non-phosphodiester internucleoside linkages.
In general, two types of oligonucleotide analogs have been reported. The first type includes those having modified phosphate linkages. The second type includes those analogs having non-phosphate internucleoside linkages. Uhlmann, E., supra.
Representative non-phosphate internucleoside linkages include siloxane, carbamate, carboxymethyl esters, acetamidate, carbonate and thioethers. Uhlmann, supra.
Of particular relevance to the present invention is the siloxane linkage or bridge.
Nucleoside dimers and hexamers having siloxane internucleoside linkages and a method of synthesizing such polymers have been reported by Ogilvie and Cormier. See, e.g., Ogilvie, K. K. and Cormier, J. F., Tetrahedron Letters, 26(35):4159-4162 (1985); Cormier J. F. and Ogilvie, K. K., Nucleic Acids Research, 16(10):4583-4594 (1988).
According to this published method, a 5'-protected nucleoside is reacted with a silylating reagent to form a 3'-silylated nucleoside, which silylated nucleoside is then reacted with a protected nucleoside to produce a fully protected, 3',5'-silyl linked dinucleoside. The fully protected, silyl linked dinucleoside is then deprotected at either terminal to carry out chain extension via another round of coupling with protected nucleosides.
Certain problems are associated with this method. When this method is employed to synthesize nucleoside polymers, the desired end product is produced in low yields ranging from about 35% to about 46%. The low yield is attributed both to the production of undesired byproducts, in particular, the 3',3'-symmetrical dimer, Uhlman, E. supra at pg. 553, resulting from self conjugation of the nucleoside building blocks, and to significant loss of useful product resulting from polymer deprotection.
The present invention provides a method of linking nucleosides with a siloxane bridge while suppressing formation of the 3',3'-dimer. This method comprises reacting a silylated nucleoside with an unprotected nucleoside in the presence of a hindered base catalyst. The use of unprotected nucelosides as compared to the procedure of Ogilvie and Cormier, has the advantages of increasing both the yield of desired end-products and the efficiency of the synthesis (thereby reducing the cost).
A preferred embodiment of the present invention, in which the reaction is carried out in the presence of a sterically hindered base catalyst, provides the additional advantage that the formation of undesired 3',3'-symmetrical dimers is minimized.